Selection of bacterial species from wastewater for potential production of poly (γ-glutamic acid): isolation, characterisation and growth kinetics

Madonsela, Ziningi

Selection of bacterial species from wastewater for potential production of poly (γ-glutamic acid): isolation, characterisation and growth kinetics

Master Thesis

2013

Abstract

Wastewater treatment plants represent a source of nutrients for microbial growth and product formation. In an approach in which bioresource productivity is maximised, it is desirable to not only achieve water treatment to the desired standard, but at the same time to harness the value in these resources. Wastewaters are a source of nutrients such as carbon, nitrogen and phosphates. Macronutrients typically comprise the major share of the operating costs of commodity bioprocesses, such as the production of alcohols, organic acids and polymers. The nutrient loads in municipal wastewaters are dilute, but add up to significant daily loads because of the massive volumes generated in urban populations. The effluent of most wastewater treatment plants in South Africa is released back into either rivers, lakes or the ocean without reuse. For many, there is a concomitant failure to comply with the country's effluent requirements. This has resulted in the need to move towards more sustainable water treatment systems, including more efficient and innovative solutions to treat wastewater. Additionally, by recovering value from waste, there is potential to improve plant operation; releasing water which is better treated, more compliant and can be reused, while simultaneously improving resource productivity and minimising environmental burden; thereby changing the economics of the wastewater treatment plant. This includes the recovery or the production of valuable resources, whilst in turn recovering clean water. Bioprocessing to reduce these nutrient loads in wastewater while producing a range of byproducts have conventionally included biogas and compost, produced with minimal modification of existing plants. In extending the potential product range of these ‘wastewater biorefineries', key design factors include using waste resources in a non-sterile environment, thus requiring a positive selection pressure for the product of interest, and ideally producing a product readily separated from the wastewater through a phase change such as precipitation. Stress and storage polymers satisfy both these requirements. In this project, we explore such a wastewater biorefinery approach in which we aim to use the nutrient component of partially treated domestic wastewater for the production of poly (γ-glutamic acid) (γPGA). γ-PGA, a polymer of D- and L-glutamic acid monomers connected by amide linkages, is a naturally occurring biopolymer, synthesized by a variety of micro-organisms. Most commonly, γ-PGA production has been studied in Bacillus species, such as B. subtilis and B. licheniformis. Bacillus species are ubiquitous in the environment, including an association with domestic wastewater treatment. Its enrichment has been associated with improved treatment processes. The favourable properties of this very promising polymer include its water solubility, anionic nature, biodegradability and non-toxicity towards humans, animals and the environment. Potential applications of γ-PGA are reported in the medical, food, cosmetic, wastewater treatment, plastic,agricultural and textile industries. In this project, we consider the production of γ-PGA by Bacillus-enriched species for the Synopsis iii partial treatment of domestic wastewater and concomitant production of the polymer for soil improvement and water treatment. To design and optimise a process using wastewater for γ-PGA production, it is important to understand the growth kinetics of Bacillus-like microorganisms that can be found in a domestic wastewater treatment plant. The base case for growth, substrate utilisation and biomass production has been presented for a Bacillus licheniformis type culture strain in this dissertation. In presenting this base case, a complete experimental methodology using both shake flasks and deep well plates is developed and appropriate analytical protocols selected. The second objective of this project was to characterise the growth kinetics of the microorganisms isolated from the wastewater obtained from the Mitchell's Plain wastewater treatment plant. Following enrichment of wastewater samples collected, 18 isolates were obtained and characterised in terms of morphology. Further, using DNA sequence data for the enzymes involved in PGA synthesis, primers were designed to identify strains carrying γ-PGA synthesis potential through molecular biology studies. Two different media; namely Medium E, containing glutamic acid, citric acid and glycerol as carbon sources, as well as a modified Medium E, in which the glutamic acid was replaced with glucose, were used for these experiments. Isolates showing reproducible growth and evidence of polymer production were selected for detailed screening in terms of growth. Based on this, six isolates were chosen. These experiments were performed in both shake flasks as well as deep well plates with the growth kinetics and biomass production by the various strains carefully analysed. Biomass concentrations varied from 2 to 8 g.L-1 while specific growth rates varied from 0.11 to 0.26 h-1 in these cultures. The results of the growth studies showed that the growth rates and biomass production of the different isolates varied greatly under identical cultivation conditions. Cultivation of the isolates in deep well plates generally yielded higher biomass concentrations in comparison to shake flask cultivation. A quantitative analysis of the data on carbon substrate utilisation in the media showed that glucose was the preferred of these, most consumed by the isolates for cell growth. Preliminary media optimisation was undertaken to identify optimal C:N:P ratios in the modified growth Medium E and to assess the impact of these medium components by using two-level Plackett-Burman factorial design. The dependence of biomass accumulation on C levels and maximum specific growth rate on N is illustrated. Response surfaces revealed the insignificant effect on the microorganism growth of varying P concentrations. Influence of P was attributed when P acted in tandem with C and N. Identification of the isolates selected for detailed screening was confirmed using 16S rRNA sequencing. There were two major families present – Proteobacteriaceae and Enterobacteriaceae – which grouped accordingly when analysed phylogenetically. The Bacillus, and opportunistically pathogenic enteric bacteria Klebsiella and Enterobacter, were reflective of genuses which have a high Synopsis iv probability of being present in a treatment plant which predominantly treats domestic wastewater containing high sewage loads, such as Mitchell's Plain. A suitable reactor configuration, capable of treating wastewater and simultaneously producing γ-PGA, was proposed for integration into Mitchell's Plain wastewater treatment plant after studying the selected Bacillus isolate in a laboratory-scale bioreactor in optimal medium. The sustained biomass production and growth rate, even at lower medium carbon inputs, shows a great potential for application by enrichment, growth and product production of this Bacillus subtilis strain in a wastewater treatment plant. Although this work was able to make a substantial contribution to the current knowledge of microbial growth kinetics in various media and bioreactor systems, the study limitations related to growth profiling in more dilute and variable nutrient concentrations, non-sterile environments and mixed culture dynamics are acknowledged. These present a scope and opportunity for further research in this exciting field. Analysis of extracted and purified γ-PGA showed a polymer composed of a combination of polysaccharides and protein (in roughly equal proportions in three of four of the isolates) in which histidine, homoserine and glutamic acid were typically the most dominant amino acids. The impure nature of this γ-PGA remains suitable for wastewater applications, but not for areas which require a product composed of specific, high molecular weight stereoisomers such as the medical industry. This study illustrates the importance of developing new experimental techniques for more γ-PGA-specific purification and improved analysis. Further it demonstrates the shortcoming of the gravimetric analysis of the crude extract typically reported in the literature. The findings of this project are intended to be used in a system which applies wastewater as an economical and sustainable source of nutrients with the aim of producing valuable products through bioprocessing applications.

Keywords

chemical engineering

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